1. Field of the Invention
The invention relates to the isolation of promoters from rice capable of directing transcription of an operably linked foreign DNA sequence preferentially in microspores and/or pollen of plants, such as rice plants. The invention also relates to the use of chimeric genes for the preferential expression of biologically active RNA of interest in microspores and/or pollen of plants, such as rice plants. Plants, such as rice plants, comprising rice pollen-preferential promoters operably linked to a foreign DNA sequence which upon transcription yield biologically active RNA preferentially in microspores and/or pollen of said plants are also provided.
2. Description of Related Art
Promoters that can provide gene expression preferentially in microspores and/or pollen of a plant and thereby provide little or no gene expression in other parts of the plant are useful in the production of transformed plants, in which a gene is to be expressed preferentially in microspores and/or pollen. The promoters can for example be used to create pollen-lethality genes and maintainer genes as described in WO 93/25695 (which is incorporated herein by reference.
A pollen-specific cDNA clone from Nicotiana tabacum, designated as NTPc303, has been isolated and characterized by Weterings et al. (1992), Plant Mol Biol 18:1101-1111. The first NTP303 transcripts are detectable on northern blot at the early bi-nucleate state and accumulate until the pollen has reached maturity. During germination and pollen tube growth in vitro new NTP303 transcripts appear. The corresponding promoter, designed NPT303 promoter, has been isolated and characterized by Weterings et al., (1995), Plant J 8:55-63. The 5′-untranslated region of the NTP303 gene was characterized by Hulzink et al., (2002), Plant Physiol 129:342-353.
A genomic clone, named Bp10, of Brassica napus has been isolated and characterized by Albani et al. (1992), Plant J 2:331-342. Bp10 contains a member of a small pollen-specific gene family. The expression of the Bp10 gene family is maximal in early bi-nucleate microspores and declines considerably in mature tri-nucleate pollen.
A pollen-specific cDNA from Zea mays inbred line W-22, designated as Zmc13, has been isolated and characterized by Hanson et al. (1989), The Plant Cell 1:173. The corresponding genomic clone, designated as Zmg13, containing substantial portions of the 5′ flanking region has been isolated and characterized by Hamilton et al. (1989), Sex Plant Reprod 2:208 (see also Hamilton et al. (1992), Plant Mol Biol 18:211). A corresponding promoter region from Zea mays inbred line H99 was isolated as described in WO 93/25695.
Three pollen-expressed genes (LAT52, LAT56 and LAT59) from tomato were characterized by McCormick et al. (1991), Symp Soc Exp Biol 45:229-244. LAT52 encodes a protein that shows amino acid sequence similarity to the protein encoded by the pollen-specific cDNA clone Zmc13 isolated from maize. The proteins encoded by LAT56 and LAT59 genes show significant sequence similarity to bacterial pectate lyases and to a fungal pectin lyase. Twell et al. (1991), Genes Dev 5:496-507, investigated the functional organization and properties of cis regulatory elements in the promoter regions of the LAT 52 and LAT59 genes that are preferentially and coordinately expressed during pollen maturation and demonstrated that only minimal (less than 200 bp) promoter proximal regions are required for developmentally regulated expression in pollen and in specific cell types of the sporophyte. They identified two upstream regions in the LAT52 promoter and further showed that a 19 bp segment from one of those regions enhanced expression of the heterologous CaMV35S promoter in pollen. Eyal et al (1995), Plant Cell 7:373-384, further identified 30 bp proximal regions of LAT 52 and LAT 59 that are essential for their expression in pollen and that confer pollen specificity when fused to the heterologous CaMV35S core promoter. Adjacent upstream elements, the 52/56 box in LAT52 and the 56/59 box in LAT59, are involved in modulating the level of expression in pollen.
Xu et al. (1999) Proc Natl Acad Shi USA 96:2554-2558 identified and characterized a gene from Lily, LGC1, which was shown to be expressed exclusively in the male gametic cells. Singh et al. (2003), FEBS Left 542:47-52, report that a 0.8 kb promoter sequence upstream of the start of the transcription site of the generative cell-specific LGC1 gene is sufficient to regulate the expression of reporter genes in a cell-specific manner and identified −242 bp as the minimal sequence necessary for male gametic cell-specific expression. In addition, a regulatory sequence required for determining generative cell-specific expression of LGC1 was identified.
Despite the fact that pollen-preferential promoters are available in the art, a need remains for alternative promoters capable of preferential expression in microspores and/or pollen of a plant, e.g. for the independent expression of several foreign DNA sequences of interest without the possibility of post-transcriptional silencing due to the use of the same promoter. In addition, the known pollen-preferential promoters, each direct a particular temporal, spatial and/or developmental expression pattern, which does not always suit particular goals. There remains thus a need for novel pollen-preferential promoters with the capacity to control transcription in microspores and/or pollen of a plant.